The Mayo Clinic Salivary Tissue-Organoid Biobanking: A Resource for Salivary Regeneration Research.

The salivary gland (SG) is an essential organ that secretes saliva, which supports versatile oral function throughout life, and is maintained by elusive epithelial stem and progenitor cells (SGSPC). Unfortunately, aging, drugs, autoimmune disorders, and cancer treatments can lead to salivary dysfunction and associated health consequences. Despite many ongoing therapeutic efforts to mediate those conditions, investigating human SGSPC is challenging due to lack of standardized tissue collection, limited tissue access, and inadequate purification methods. Herein, we established a diverse and clinically annotated salivary regenerative biobanking at the Mayo Clinic, optimizing viable salivary cell isolation and clonal assays in both 2D and 3D-matrigel growth environments. Our analysis identified ductal epithelial cells in vitro enriched with SGSPC expressing the CD24/EpCAM/CD49f+ and PSMA- phenotype. We identified PSMA expression as a reliable SGSPC differentiation marker. Moreover, we identified progenitor cell types with shared phenotypes exhibiting three distinct clonal patterns of salivary differentiation in a 2D environment. Leveraging innovative label-free unbiased LC-MS/MS-based single-cell proteomics, we identified 819 proteins across 71 single cell proteome datasets from purified progenitor-enriched parotid gland (PG) and sub-mandibular gland (SMG) cultures. We identified distinctive co-expression of proteins, such as KRT1/5/13/14/15/17/23/76 and 79, exclusively observed in rare, scattered salivary ductal basal cells, indicating the potential de novo source of SGSPC. We also identified an entire class of peroxiredoxin peroxidases, enriched in PG than SMG, and attendant H2O2-dependent cell proliferation in vitro suggesting a potential role for PRDX-dependent floodgate oxidative signaling in salivary homeostasis. The distinctive clinical resources and research insights presented here offer a foundation for exploring personalized regenerative medicine.

Genetic variation in the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain.

Bacteria harboring conjugative plasmids have the potential for spreading antibiotic resistance through horizontal gene transfer. It is described that the selection and dissemination of antibiotic resistance is enhanced by stressors, like metals or antibiotics, which can occur as environmental contaminants. This study aimed at unveiling the composition of the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain (H1FC54) under different mating conditions. To meet this objective, plasmid pulsed field gel electrophoresis, optical mapping analyses and DNA sequencing were used in combination with phenotype analysis. Strain H1FC54 was observed to harbor five plasmids, three of which were conjugative and two of these, pH1FC54_330 and pH1FC54_140, contained metal and antibiotic resistance genes. Transconjugants obtained in the absence or presence of tellurite (0.5 µM or 5 µM), arsenite (0.5 µM, 5 µM or 15 µM) or ceftazidime (10 mg/L) and selected in the presence of sodium azide (100 mg/L) and tetracycline (16 mg/L) presented distinct phenotypes, associated with the acquisition of different plasmid combinations, including two co-integrate plasmids, of 310 kbp and 517 kbp. The variable composition of the conjugative plasmidome, the formation of co-integrates during conjugation, as well as the transfer of non-transferable plasmids via co-integration, and the possible association between antibiotic, arsenite and tellurite tolerance was demonstrated. These evidences bring interesting insights into the comprehension of the molecular and physiological mechanisms that underlie antibiotic resistance propagation in the environment.

Quinolone resistant Aeromonas spp. as carriers and potential tracers of acquired antibiotic resistance in hospital and municipal wastewater.

Members of the genus Aeromonas are recognized carriers of antibiotic resistance in aquatic environments. However, their importance on the spread of resistance from hospital effluents to the environment is poorly understood. Quinolone resistant Aeromonas spp. (n = 112) isolated from hospital effluent (HE) and from raw (RWW) and treated wastewater (TWW) of the receiving urban wastewater treatment plant (UWTP) were characterized. Species identification and genetic intraspecies diversity were assessed based on the 16S rRNA, cpn60 and gyrB genes sequence analysis. The antibiotic resistance phenotypes and genotypes (qnrA, qnrB, qnrC, qnrD, qnrS, qnrVC; qepA; oqxAB; aac(6')-Ib-cr; blaOXA; incU) were analyzed in function of the origin and taxonomic group. Most isolates belonged to the species Aeromonas caviae and Aeromonas hydrophila (50% and 41%, respectively). The quinolone and the beta-lactamase resistance genes aac(6')-Ib-cr and blaOXA, including gene blaOXA-101, identified for the first time in Aeromonas spp., were detected in 58% and 56% of the isolates, respectively, with identical prevalence in HE and UWTP wastewater. In contrast, the gene qnrS2 was observed mainly in isolates from the UWTP (51%) and rarely in HE isolates (3%), suggesting that its origin is not the clinical setting. Bacterial groups and genes that allow the identification of major routes of antibiotic resistance dissemination are valuable tools to control this problem. In this study, it was concluded that members of the genus Aeromonas harboring the genes aac(6')-Ib-cr and blaOXA are relevant tracers of antibiotic resistance dissemination in wastewater habitats, while those yielding the gene qnrS2 allow the traceability from non-clinical sources.

Multidrug Resistance in Quinolone-Resistant Gram-Negative Bacteria Isolated from Hospital Effluent and the Municipal Wastewater Treatment Plant.

This study is aimed to assess if hospital effluents represent an important supplier of multidrug-resistant (MDR) Gram-negative bacteria that, being discharged in the municipal collector, may be disseminated in the environment and bypassed in water quality control systems. From a set of 101 non-Escherichia coli Gram-negative bacteria with reduced susceptibility to quinolones, was selected a group of isolates comprised by those with the highest indices of MDR (defined as nonsusceptibility to at least one agent in six or more antimicrobial categories, MDR ≥6) or resistance to meropenem or ceftazidime (n = 25). The isolates were identified and characterized for antibiotic resistance phenotype, plasmid-mediated quinolone resistance (PMQR) genes, and other genetic elements and conjugative capacity. The isolates with highest MDR indices were mainly from hospital effluent and comprised ubiquitous bacterial groups of the class Gammaproteobacteria, of the genera Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Klebsiella, and Pseudomonas, and of the class Flavobacteriia, of the genera Chryseobacterium and Myroides. In this group of 25 strains, 19 identified as Gammaproteobacteria harbored at least one PMQR gene (aac(6')-Ib-cr, qnrB, qnrS, or oqxAB) or a class 1 integron gene cassette encoding aminoglycoside, sulfonamide, or carbapenem resistance. Most of the E. coli J53 transconjugants with acquired antibiotic resistance resulted from conjugation with Enterobacteriaceae. These transconjugants demonstrated acquired resistance to a maximum of five classes of antibiotics, one or more PMQR genes and/or a class 1 integron gene cassette. This study shows that ubiquitous bacteria, other than those monitored in water quality controls, are important vectors of antibiotic resistance and can be disseminated from hospital effluent to aquatic environments. This information is relevant to support management options aiming at the control of this public health problem.

Genetic characterization of fluoroquinolone resistant Escherichia coli from urban streams and municipal and hospital effluents.

Escherichia coli with reduced susceptibility to ciprofloxacin, isolated from urban streams, wastewater treatment plants and hospital effluent between 2004 and 2012, were compared based on multilocus sequence typing (MLST), quinolone and beta-lactam resistance determinants and plasmid replicon type. Isolates from the different types of water and isolation dates clustered together, suggesting the persistence and capacity to propagate across distinct aquatic environments. The most prevalent MLST groups were ST10 complex and ST131. Almost all isolates (98%) carried mutations in the chromosomal genes gyrA and/or parC, and 10% possessed the genes qepA, aac(6('))-Ib-cr and/or qnrS1. Over 80% of the isolates were resistant to three or more classes of antibiotics (MDR ≥ 3). The most prevalent beta-lactamase encoding gene was blaTEM, followed by blaCTX-M-15, co-existing with plasmid mediated quinolone resistance. The plasmid replicon types of the group IncF were the most prevalent and distributed by different MLST groups. The genes aac(6('))-Ib-cr and/or qnrS1 could be transferred by conjugation in combination with the genes blaTEM,blaSHV-12 or blaOXA-1 and the plasmid replicon types I1-Iγ, K, HI2 and/or B/O. The potential of multidrug resistant E. coli with reduced susceptibility to ciprofloxacin, harboring mobile genetic elements and with ability to conjugate and transfer resistance genes, to spread and persist across different aquatic environments was demonstrated.

Molecular evidence of the close relatedness of clinical, gull and wastewater isolates of quinolone-resistant Escherichia coli.

Escherichia coli with reduced susceptibility to quinolones isolated from different environmental sources (urban wastewater treatment plants, n=61; hospital effluent, n=10; urban streams, n=9; gulls, n=18; birds of prey, n=17) and from hospitalised patients (n=28) were compared based on multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The habitats with the most diversified genotypes of quinolone-resistant E. coli, corresponding to the highest genetic diversity (H'), were wastewater and gulls. In addition, genetically distinct populations were observed in clinical samples and birds of prey, suggesting the influence of the habitat or selective pressures on quinolone-resistant E. coli. The close genetic relatedness between isolates of clinical origin and from gulls and wastewater suggests the existence of potential routes of propagation between these sources. The most common sequence types were ST131 and ST10, with ST131 being highly specific to patients, although distributed in all of the other habitats except birds of prey. The prevalence of antimicrobial resistance was significantly higher in isolates from patients and gulls than from other sources (P<0.01), suggesting that the effect of selective pressures met by isolates subjected to strong human impacts. The evidence presented suggests the potential circulation of bacteria between the environmental and clinical compartments, with gulls being a relevant vector of bacteria and resistance genes.

Insights into the relationship between antimicrobial residues and bacterial populations in a hospital-urban wastewater treatment plant system.

The relationship between antimicrobial residues, antibiotic resistance prevalence and bacterial community composition in hospital effluent and in the receiving wastewater treatment plant was studied. Samples from hospital effluent, raw inflow and final effluent of the receiving wastewater treatment plant were characterized for amoxicillin and ciprofloxacin resistance prevalence, content of heavy metals and antimicrobial residues and bacterial community structure, based on 16S rRNA gene PCR-DGGE analysis. The concentration of fluoroquinolones, arsenic and mercury was in general higher in hospital effluent than in raw inflow, while the opposite was observed for tetracyclines, sulfonamides and penicillin G. The prevalence of ciprofloxacin resistance was significantly higher in hospital effluent than in raw inflow. The concentration of antimicrobial residues was observed to be significantly correlated with the prevalence of antibiotic resistant bacteria and with variations in the bacterial community. Hospital effluent was confirmed as a relevant, although not unique, source of antimicrobial residues and antibiotic resistant bacteria to the wastewater treatment plant. Moreover, given the high loads of antibiotic residues and antibiotic resistant bacteria that may occur in hospital effluents, these wastewater habitats may represent useful models to study and predict the impact of antibiotic residues on bacterial communities.

Quinolone-resistant Escherichia coli isolated from birds of prey in Portugal are genetically distinct from those isolated from water environments and gulls in Portugal, Spain and Sweden.

The influence of geographic distribution and type of habitat on the molecular epidemiology of ciprofloxacin resistant Escherichia coli was investigated. Ciprofloxacin resistant E. coli from wastewater, urban water with faecal contamination and faeces of gulls, pigeons and birds of prey, from Portugal, Spain and Sweden were compared based on multi-locus sequence typing (MLST) and quinolone resistance genetic determinants. Multi-locus sequence typing allowed the differentiation of E. coli lineages associated with birds of prey from those inhabiting gulls and waters. E. coli lineages of clinical relevance, such as the complex ST131, were detected in wastewater, streams and gulls in Portugal, Spain and Sweden. Quinolone resistance was due to gyrA and parC mutations, although distinct mutations were detected in birds of prey and in wastewater, streams and gulls isolates. These differences were correlated with specific MLST lineages, suggesting resistance inheritance. Among the plasmid-mediated quinolone resistance genes, only aac(6')-ib-cr and qnrS were detected in wastewater, streams and gulls isolates, but not in birds of prey. The horizontal transfer of the gene aac(6')-ib-cr could be inferred from its occurrence in different MLST lineages.

Vancomycin resistant enterococci: from the hospital effluent to the urban wastewater treatment plant.

Vancomycin is an important antibiotic to treat serious nosocomial enterococci infections. Human activities, in particular those related with clinical practices performed in hospitals, can potentiate the transfer and selection of clinically-relevant resistant bacteria such as vancomycin resistant enterococci (VRE). Indeed, previous studies demonstrated the occurrence of VRE in urban wastewater treatment plants and related environments (e.g. sewage, rivers). In this study, the occurrence of VRE in a hospital effluent and in the receiving urban wastewater treatment plant was investigated. Vancomycin and ciprofloxacin resistant bacteria occurred in the hospital effluent and in raw municipal inflow at densities of 10(3) to 10(2) CFU mL(-1), being significantly more prevalent in the hospital effluent than in the urban wastewater. Most of the VRE isolated from the hospital effluent belonged to the species Enterococcus faecalis and Enterococcus faecium and presented multidrug-resistance phenotypes to ciprofloxacin, tetracycline, erythromycin, and high-level gentamicin. The same pattern was observed in clinical isolates and in enterococci isolated from the final effluent of the urban wastewater treatment plant. These results show that hospital effluents discharged into urban wastewater treatment plants may be a relevant source of resistance spread to the environment.

Human health implications of clinically relevant bacteria in wastewater habitats.

The objective of this review is to reflect on the multiple roles of bacteria in wastewater habitats with particular emphasis on their harmful potential for human health. Indigenous bacteria promote a series of biochemical and metabolic transformations indispensable to achieve wastewater treatment. Some of these bacteria may be pathogenic or harbour antibiotic resistance or virulence genes harmful for human health. Several chemical contaminants (heavy metals, disinfectants and antibiotics) may select these bacteria or their genes. Worldwide studies show that treated wastewater contain antibiotic resistant bacteria or genes encoding virulence or antimicrobial resistance, evidencing that treatment processes may fail to remove efficiently these bio-pollutants. The contamination of the surrounding environment, such as rivers or lakes receiving such effluents, is also documented in several studies. The current state of the art suggests that only some of antibiotic resistance and virulence potential in wastewater is known. Moreover, wastewater habitats may favour the evolution and dissemination of new resistance and virulence genes and the emergence of new pathogens. For these reasons, additional research is needed in order to obtain a more detailed assessment of the long-term effects of wastewater discharges. In particular, it is important to measure the human and environmental health risks associated with wastewater reuse.